Neurotrophic actions of novel compounds designed from cyclopentenone prostaglandins

Previously we found that some cyclopentenone prostaglandin derivatives promoted neurite outgrowth from PC12 cells and dorsal root ganglia explants in the presence of nerve growth factor; and so we referred to them as neurite outgrowth-promoting prostaglandins (NEPPs). In this study, NEPPs protected HT22 cells against oxidative glutamate toxicity. NEPP6, one of the most effective promoters of neurite outgrowth in PC12 cells, protected the cells most potently among NEPPs 1--10. Several derivatives, NEPPs 11--19, were newly synthesized based on the chemical structure of NEPP6. NEPP11 had a more potent neuroprotective effect than NEPP6. NEPP11 also prevented the death of cortical neurons induced by various stimuli and reduced ischemic brain damage in mice. Biotinylated compounds of NEPPs were synthesized to investigate their cellular accumulation. NEPP6-biotin protected the cells and emitted potent signals from the cells. In contrast, biotinylated non-neuroprotective derivatives emitted much weaker signals. These results suggest that NEPPs are novel types of neurotrophic compounds characterized by their dual biological activities of promoting neurite outgrowth and preventing neuronal death and that their accumulation in the cells is closely associated with their neuroprotective actions.     

Reconstitution of interactions of Murine gammaherpesvirus 68 M11 with Bcl-2 family proteins in yeast

One of the mechanisms of defense against viral infection is induction of apoptosis in infected cells. To escape this line of protection, genomes of many viruses encode for proteins that inhibit apoptosis. Murid herpesvirus 4 gene M11 encodes for homologue of cellular Bcl-2 proteins that inhibits apoptosis and autophagy in infected cell. To study a role of M11 in regulation of apoptosis we have established a yeast model system in which the action of M11 together with proapoptotic proteins Bax, Bak and Bid can be studied. When expressed in yeast, M11 did not inhibit autophagic pathway, so only effects of expression of M11 on activity of coexpressed proapoptotic proteins could be observed. In this experimental setting M11 potently inhibited both proapoptotic multidomain proteins Bax and Bak. The antiapoptotic activity of M11 was suppressed by coexpression of proapoptotic BH3-only protein tBid, indicating that M11 inhibits apoptosis likely by the same mechanism as cellular antiapoptotic proteins Bcl-2 or Bcl-XL.     

Synthesis and biological evaluation of sulfonyl acrylonitriles as novel inhibitors to peritoneal carcinomatosis

The vast majority of cancer patients die from metastasis, the process by which cancer cells spread to secondary tissues through body fluids. Peritoneal carcinomatosis is a type of metastasis in which cancer cells gain access to the intra-abdominal cavity and then implant in the peritoneum, the thin tissue that lines the abdominal wall and internal organs. Unfortunately, peritoneal carcinomatosis can occur following surgical resection of intra-abdominal malignancies. We previously reported proapoptotic activity of (2E)-3-[[4-(1,1-dimethylethyl)phenyl]sulfonyl]-2-propenenitrile (BAY 11-7085, 1) on colon and pancreatic cancer cells during adhesion and demonstrated that this compound could significantly inhibit peritoneal carcinomatosis in mice.(1,2) In order to determine the chemical basis of the anti-metastatic properties of BAY 11-7085, a series of analogs were synthesized and evaluated for their ability to induce apoptosis in pancreatic and ovarian cancer cells during adhesion to mesothelial cells, which line the surface of the peritoneum. The co-culture assay results were validated using a murine peritoneal carcinomatosis model. These analogs may greatly benefit patients undergoing surgical resections of colorectal, pancreatic, and ovarian cancers depending on their tolerability.     

Induction of cancer cell apoptosis by alpha-tocopheryl succinate: molecular pathways and structural requirements.

The vitamin E analog alpha-tocopheryl succinate (alpha-TOS) can induce apoptosis. We show that the proapoptotic activity of alpha-TOS in hematopoietic and cancer cell lines involves inhibition of protein kinase C (PKC), since phorbol myristyl acetate prevented alpha-TOS-triggered apoptosis. More selective effectors indicated that alpha-TOS reduced PKCalpha isotype activity by increasing protein phosphatase 2A (PP2A) activity. The role of PKCalpha inhibition in alpha-TOS-induced apoptosis was confirmed using antisense oligonucleotides or PKCalpha overexpression. Gain- or loss-of-function bcl-2 mutants implied modulation of bcl-2 activity by PKC/PP2A as a mitochondrial target of alpha-TOS-induced proapoptotic signals. Structural analogs revealed that alpha-tocopheryl and succinyl moieties are both required for maximizing these effects. In mice with colon cancer xenografts, alpha-TOS suppressed tumor growth by 80%. This epitomizes cancer cell killing by a pharmacologically relevant compound without known side effects.     

Overlapping functions of microRNAs in control of apoptosis during Drosophila embryogenesis

Regulation of apoptosis is crucial for tissue homeostasis under normal development and environmental stress. In Drosophila, cell death occurs in different developmental processes including embryogenesis. Here, we report that two members of the miR-2 seed family of microRNAs, miR-6 and miR-11, function together to limit the level of apoptosis during Drosophila embryonic development. Mutants lacking both miR-6 and miR-11 show embryonic lethality and defects in the central nervous system (CNS). We provide evidence that miR-6/11 functions through regulation of the proapoptotic genes, reaper (rpr), head involution defective (hid), grim and sickle (skl). Upregulation of these proapoptotic genes is responsible for the elevated apoptosis and the CNS defects in the mutants. These findings demonstrate that the activity of the proapoptotic genes is kept in check by miR-6/11 to ensure normal development.     

Antiapoptotic and proapoptotic action of various amino acids and analogs in starving MOLT-4 cells.

This study is based on our previous findings showing that certain amino acids may protect hybridoma cells against starvation-induced apoptosis. In the present work we have screened 44 amino acids and analogs for their capacity of modulating apoptosis in human T-lymphoblastic leukemia cell line MOLT-4 exposed to starvation in a nutrient-poor medium. The panel of tested substances was found to contain not only compounds with antiapoptotic activity (e.g., l-glutamine, l-histidine, glycine, l-proline, and l-2-aminopentanoic acid), but also compounds with proapoptotic activity (e.g., l-phenylalanine, l-tryptophan, l-arginine, and l-2-aminohexanoic acid). The apoptosis-modulating effects were dependent on fine details of the structure of the compounds. A switch from antiapoptotic activity to proapoptotic activity was found between 6-aminohexanoic acid and 7-aminoheptanoic acid, as well as between l-2-aminopentanoic acid and l-2-aminohexanoic acid. D-amino acids tested were without effect.     

Caspase-8 gene transduction augments radiation-induced apoptosis in DLD-1 cells

Caspase-8 is a member of the cysteine protease family that plays a critical role in death receptor-mediated apoptosis. We previously demonstrated that adenovirally transduced caspase-8 efficiently induced apoptosis in tumor cells (Shinoura et al. (2000) Hum. Gene Ther. 11, 1123-1137). However, to ensure safety in clinical applications some devise for minimization of the dose of adenoviral vector required for sufficient antitumor effect is needed. In this study, we evaluated the proapoptotic effect in DLD-1 colon cancer cells of a combination of low-dose infection with an adenoviral vector expressing caspase-8 and X-ray irradiation. Under these conditions, X-ray irradiation strongly induced apoptosis whereas irradiation without transduction only had a trace proapoptotic effect. Overexpression of bcl-xL strongly blocked the activation of caspase-8 and induction of apoptosis, suggesting that adenovirally transduced caspase-8 was activated at a point downstream of mitochondria. This combination strategy may be a useful modality for gene therapy of colorectal cancer.     

In transduced cells, the US3 protein kinase of herpes simplex virus 1 precludes activation and induction of apoptosis by transfected procaspase 3

The US3 protein kinase of herpes simplex virus 1 blocks apoptosis induced by replication-incompetent virus mutants, proapoptotic members of the Bcl-2 family of proteins, and by a variety of other agents that act at the premitochondrial level in the proapoptotic cascade. To define the role of US3 in blocking apoptosis at the postmitochondrial level, we investigated the US3 protein kinase in transduced cells that were either transfected with a plasmid encoding procaspase 3 or superinfected with a proapoptotic mutant virus lacking the gene encoding the infected cell protein no. 4. (i) We show that US3 blocks the proteolytic cleavage that generates active caspase 3 from the transfected zymogen procaspase 3, concomitant with inhibition of apoptosis. (ii) Studies based on detection of fluorescence emitted upon cleavage of a synthetic caspase 3 substrate showed that expression of the US3 kinase and appearance of the cleaved substrate were mutually exclusive. (iii) An affinity-purified glutathione S-transferase (GST)-US3 fusion protein, but not the inactive GST-US3(K220N) protein, phosphorylated procaspase 3 in vitro. The studies published earlier on the effect of US3 on the upstream regulatory proteins and current studies suggest that the US3 protein kinase may act on several proteins in the proapoptotic cascade to enable the virus to complete its replication.     

Viral proteins targeting mitochondria: controlling cell death

Mitochondrial membrane permeabilization (MMP) is a critical step regulating apoptosis. Viruses have evolved multiple strategies to modulate apoptosis for their own benefit. Thus, many viruses code for proteins that act on mitochondria and control apoptosis of infected cells. Viral proapoptotic proteins translocate to mitochondrial membranes and induce MMP, which is often accompanied by mitochondrial swelling and fragmentation. From a structural point of view, all the viral proapoptotic proteins discovered so far contain amphipathic alpha-helices that are necessary for the proapoptotic effects and seem to have pore-forming properties, as it has been shown for Vpr from human immunodeficiency virus-1 (HIV-1) and HBx from hepatitis B virus (HBV). In contrast, antiapoptotic viral proteins (e.g., M11L from myxoma virus, F1L from vaccinia virus and BHRF1 from Epstein-Barr virus) contain mitochondrial targeting sequences (MTS) in their C-terminus that are homologous to tail-anchoring domains. These domains are similar to those present in many proteins of the Bcl-2 family and are responsible for inserting the protein in the outer mitochondrial membrane leaving the N-terminus of the protein facing the cytosol. The antiapoptotic proteins K7 and K15 from avian encephalomyelitis virus (AEV) and viral mitochondria inhibitor of apoptosis (vMIA) from cytomegalovirus are capable of binding host-specific apoptosis-modulatory proteins such as Bax, Bcl-2, activated caspase 3, CAML, CIDE-B and HAX. In conclusion, viruses modulate apoptosis at the mitochondrial level by multiple different strategies.     

Differential roles for alpha(M)beta(2) integrin clustering or activation in the control of apoptosis via regulation of akt and ERK survival mechanisms

The role of integrins in leukocyte apoptosis is unclear, some studies suggest enhancement, others inhibition. We have found that beta(2)-integrin engagement on neutrophils can either inhibit or enhance apoptosis depending on the activation state of the integrin and the presence of proapoptotic stimuli. Both clustering and activation of alpha(M)beta(2) delays spontaneous, or unstimulated, apoptosis, maintains mitochondrial membrane potential, and prevents cytochrome c release. In contrast, in the presence of proapoptotic stimuli, such as Fas ligation, TNFalpha, or UV irradiation, ligation of active alpha(M)beta(2) resulted in enhanced mitochondrial changes and apoptosis. Clustering of inactive integrins did not show this proapoptotic effect and continued to inhibit apoptosis. This discrepancy was attributed to differential signaling in response to integrin clustering versus activation. Clustered, inactive alpha(M)beta(2) was capable of stimulating the kinases ERK and Akt. Activated alpha(M)beta(2) stimulated Akt, but not ERK. When proapoptotic stimuli were combined with either alpha(M)beta(2) clustering or activation, Akt activity was blocked, allowing integrin activation to enhance apoptosis. Clustered, inactive alpha(M)beta(2) continued to inhibit stimulated apoptosis due to maintained ERK activity. Therefore, beta(2)-integrin engagement can both delay and enhance apoptosis in the same cell, suggesting that integrins can play a dual role in the apoptotic progression of leukocytes.     

Identification of X-linked inhibitor of apoptosis-associated factor-1 as an interferon-stimulated gene that augments TRAIL Apo2L-induced apoptosis

In the course of gene array studies aimed at identifying IFN-stimulated genes associated with interferon beta (IFN-beta)-induced apoptosis, we identified X-linked inhibitor of apoptosis-associated factor-1 (XAF1) as a novel IFN-stimulated gene. XAF1 mRNA was up-regulated by IFN-alpha and IFN-beta in all cells examined. However, IFNs induced high levels of XAF1 protein predominantly in cell lines sensitive to the proapoptotic effects of IFN-beta. In apoptosis-resistant cells including WM164 melanoma, WM35 melanoma, U937 pro-monocytic leukemia, and HT1080 fibrosarcoma cells, XAF1 mRNA was strongly up-regulated but XAF1 protein was up-regulated only weakly or not at all. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a critical mediator of IFN-beta-induced apoptosis, but most melanoma cell lines were resistant to recombinant TRAIL protein. For example, A375 melanoma cells were defective in TRAIL induction by IFN-beta and were resistant to TRAIL-induced apoptosis. However, IFN-beta pretreatment sensitized them to subsequent recombinant TRAIL-induced apoptosis. A375 cells expressing XAF1 constitutively were more sensitive to TRAIL-induced apoptosis compared with empty vector-transfected cells. The degree of sensitization by XAF1 was similar to that provided by IFN pretreatment and was correlated with the level of XAF1 expressed. Furthermore, the overexpression of the zinc-finger portion of XAF1 blocked IFN-dependent sensitization of A375 melanoma cells to the proapoptotic effects of TRAIL. These results suggested that IFN-dependent induction of XAF1 strongly influenced cellular sensitivity to the proapoptotic actions of TRAIL.     

Induction of apoptosis by alkaloids, non-protein amino acids, and cardiac glycosides in human promyelotic HL-60 cells

The induction of apoptosis by 66 alkaloids of the quinoline, quinolizidine, pyrrolizidine, isoquinoline, indole, terpene, tropane, steroid, purine, and piperidine type, of 9 cardiac glycosides, 11 non-protein amino acids and 10 further secondary metabolites was assayed in HL-60 cell cultures and measured by quantification of the subdiploid DNA content by flow cytometry, detection of DNA fragmentation by gel electrophoresis, and cell morphology. Several alkaloids of the isoquinoline, quinoline, and indole type were active, whereas quinol-izidine, tropane, pyrrolizidine, terpene and piperdine alkaloids were mostly inactive. The proapoptotic alkaloids can be characterized by their property to inhibit protein biosynthesis and their intercalation into DNA at the same time, or by their inhibition of microtubule formation. All cardiac glycosides, which are both membrane detergents and Na+,K+-ATPase inhibitors, are potent apoptosis inducers. Also proapoptotic were a few non-protein amino acids, podophyllotoxin and the flavonoid quercetin.     

New bichalcone analogs as NF-κB inhibitors and as cytotoxic agents inducing Fas/CD95-dependent apoptosis

A series of novel bichalcone analogs were synthesized and evaluated in lipopolysaccharide (LPS)-activated microglial cells as inhibitors of nitric oxide (NO) and for in vitro anticancer activity using a limited panel of four human cancer cell lines. All analogs inhibited NO production. Compounds 4 and 11 exhibited optimal activity with IC(50) values of 0.3 and 0.5 μM, respectively, and were at least 38-fold better than the positive control. A mechanism of action study showed that both compounds significantly blocked the nuclear translocation of NF-κB p65 and up-regulation of iNOS at 1.0 μM. Compound 4 and three other analogs (3, 20, and 23) exerted significant in vitro anticancer activity GI(50) values ranging from 0.70 to 13.10 μM. A mode of action study using HT-29 colon cancer cells showed that 23 acts by inducing apoptosis signaling.     

Bak but not Bax is essential for Bcl-xS-induced apoptosis

Bcl-x(S), a proapoptotic member of the Bcl-2 protein family, is localized in the mitochondria and induces apoptosis in a caspase- and BH3-dependent manner by a mechanism involving cytochrome c release. The way in which Bcl-x(S) induces caspase activation and cytochrome c release, as well as the relationship between Bcl-x(S) and other proapoptotic members of the Bcl-2 family, is not known. Here we used embryonic fibroblasts derived from mice deficient in the multidomain proapoptotic members of the Bcl-2 family (Bax and Bak) and the apoptotic components of the apoptosome (Apaf-1 and caspase-9) to unravel the cascade of events by which Bcl-x(S) promotes apoptosis. Our results show that Bak but not Bax is essential for Bcl-x(S)-induced apoptosis. Bcl-x(S) induced activation of Bak, which in turn promoted apoptosis by apoptosome-dependent and -independent pathways. These findings provide the first evidence that a proapoptotic Bcl-2 family protein induces apoptosis exclusively via Bak.     

Phosphorylation of BCL-2 regulates ER Ca2+ homeostasis and apoptosis

Phosphorylation of BCL-2 within an unstructured loop inhibits its antiapoptotic effect. We found that phosphorylated BCL-2 predominantly localized to the endoplasmic reticulum (ER) and tested whether phosphorylation would control its activity at this organelle, where Ca(2+) dynamics serve as a critical control point for apoptosis. Phosphorylation greatly inhibits the ability of BCL-2 to lower [Ca(2+)](er) and protect against Ca(2+)-dependent death stimuli. Cells expressing nonphosphorylatable BCL-2(AAA) exhibited increased leak of Ca(2+) from the ER and further diminished steady-state [Ca(2+)](er) stores when compared to cells expressing BCL-2(wt). Consequently, when BCL-2 is phosphorylated, Ca(2+) discharge from the ER is increased, with a secondary increase in mitochondrial Ca(2+) uptake. We also demonstrate that phosphorylation of BCL-2 inhibits its binding to proapoptotic family members. This inhibitory mechanism manifested at the ER, where phosphorylated BCL-2 was unable to bind proapoptotic members. [Ca(2+)](er) proved coordinate with the capacity of BCL-2 to bind proapoptotic BH3-only members, further integrating the apoptotic pathway and Ca(2+) modulation. Unexpectedly, the regulation of ER Ca(2+) dynamics is a principal avenue whereby BCL-2 phosphorylation alters susceptibility to apoptosis.     

Thr-370 is responsible for CDK11(p58) autophosphorylation, dimerization, and kinase activity

CDK11(p58), a member of the p34(cdc2)-related kinase family, is associated with cell cycle progression, tumorigenesis, and proapoptotic signaling. It is also required for the maintenance of chromosome cohesion, the maturation of centrosome, the formation of bipolar spindle, and the completion of mitosis. Here we identified that CDK11(p58) interacted with itself to form homodimers in cells, whereas D224N, the kinase-dead mutant, failed to form homodimers. CDK11(p58) was autophosphorylated, and the main functions of CDK11(p58), such as kinase activity, transactivation of nuclear receptors, and proapoptotic signal transduction, were dependent on its autophosphorylation. Furthermore, the in vitro kinase assay indicated that CDK11(p58) was autophosphorylated at Thr-370. By mutagenesis, we created CDK11(p58) T370A and CDK11(p58) T370D, which mimic the dephosphorylated and phosphorylated forms of CDK11(p58), respectively. The T370A mutant could not form dimers and be phosphorylated by the wild type CDK11(p58) and finally lost the kinase activity. Further functional research revealed that T370A failed to repress the transactivation of androgen receptor and enhance the cell apoptosis. Overall, our data indicated that Thr-370 is responsible for the autophosphorylation, dimerization, and kinase activity of CDK11(p58). Moreover, Thr-370 mutants might affect CDK11(p58)-mediated signaling pathways.     

Inhibition of anti-apoptotic signals by Wortmannin induces apoptosis in the remote myocardium after LAD ligation: evidence for a protein kinase C-δ-dependent pathway

It has been shown that, in the remote myocardium after infarction (MI), protein kinase C (PKC) inhibition reduces apoptosis both by blocking proapoptotic pathways and by activating antiapoptotic signals including the Akt pathway. However, it was open if vice versa, blockade of antiapoptotic pathways may influence proapoptotic signals. To clarify this, the present study tested the effects of the PI3-kinase blocker Wortmannin on proapoptotic signals and on apoptosis execution in the remote myocardium after infarction. Rats were subjected to MI by LAD ligation in situ. Some were pre-treated with Wortmannin alone or in combination with the PKC inhibitor Chelerythrine. After 24 h, pro- and anti-apoptotic signals (caspase-3, PKC isoforms, p38-MAPK, p42/44-MAPK, Akt, Bad), and marker of apoptosis execution (TUNEL) were quantified in the myocardium remote from the infarction. Wortmannin treatment increased apoptosis in the remote myocardium both at baseline and after MI, together with an activation of the PKC-δ/p38-MAPK-pathway. PKC-ε and p42/44-MAPK were unaffected. Combined treatment with Wortmannin and Chelerythrine fully reversed the pro-apoptotic effects of Wortmannin both at baseline and after MI. The PKC-δ-p38-MAPK-pathway as a strong signal for apoptosis in the non-infarcted myocardium can be influenced by targeting the anti-apoptotic PI3-kinase pathway. This gives evidence of a bi-directional crosstalk of pro- and anti-apoptotic signals after infarction.     

Transforming growth factor-beta 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells

Transforming growth factor-beta1 (TGF-beta1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-beta1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-beta1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-beta1 induced apoptosis. The proapoptotic effects of TGF-beta1 occur via a p53-independent pathway, since p53(null) FDCP-Mix demonstrated the same responses to TGF-beta1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-x(L) expression. In FDCP-Mix cells, neither IL-3 nor TGF-beta1 induced any change in Bcl-x(L) protein levels or the proapoptotic proteins Bad or Bax. However, TGF-beta1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-beta1-induced apoptosis but was incapable of inhibiting TGF-beta1-mediated growth arrest. We conclude that TGF-beta1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-x(L). The significance of these results for stem cell survival in bone marrow are discussed.